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Braz J Med Biol Res 31(11) 1998 Macrophage cytotoxicity and mycoplasma
Thioglycollate-elicited murine
macrophages are cytotoxic to
Mycoplasma arginini-infected
YAC-1 tumor cells
Departamentos de 1Imunologia and 2Microbiologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, São Paulo, SP, Brasil F. Ribeiro-Dias1, M. Russo1,
F.R.F. Nascimento1,
J.A.M. Barbuto1,
J. Timenetsky2 and S. Jancar1
Abstract
Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elic-ited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited mac-rophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 ± 8.6%) and low production of NO (4.7 ± 3.1
µM NO2
-). These macrophages co-cultured with
mycoplasma-in-fected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ± 9.1%; P<0.05) and higher NO production (48.5 ± 13 µM NO2-; P<0.05). Addition of L-NAME (10 mM), an inhibitor of NO
synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 ± 2% (P<0.05) and NO production to 3 ± 4 µM NO2- (P<0.05). The
present data show that Mycoplasma arginini is able to induce macro-phage cytotoxic activity and that this activity is partially mediated by NO.
Correspondence
S. Jancar
Departamento de Imunologia ICB, USP
Av. Prof. Lineu Prestes, 2415 05508-900 São Paulo, SP Brasil
Fax: +55-11-818-7224
E-mail: sojancar@biomed.icb2.usp.br Presented at the XIII Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brasil, August 26-29, 1998. Research supported by FAPESP (No. 95/9336-1). Received April 15, 1998 Accepted August 10, 1998 Key words •Macrophages •Cytotoxicity •Mycoplasma •Tumor cells •Nitric oxide
Macrophages are usually found in tumor infiltrates where they exert cytostatic/cyto-toxic activities against tumor cells. The tumoricidal activity is enhanced by activa-tion of macrophages with bacterial products or cytokines (1,2). Recently nitric oxide (NO) has been indicated as a critical effector mol-ecule for macrophage anti-tumor activity (3,4). Macrophages can be induced to re-lease NO upon stimulation with a variety of
stimuli such as bacterial products or cyto-kines (3,5). More recently it has been re-ported that mycoplasma-treated macrophag-es release large amounts of NO (6).
YAC-1 tumor cells have been classically used as targets for natural killer (NK) cells. Resident macrophages do not present anti-YAC-1 activity, but lymphokine-activated macrophages are able to kill YAC-1 cells (7). The mechanism by which
lymphokine-Brazilian Journal of Medical and Biological Research (1998) 31: 1425-1428 ISSN 0100-879X Short Communication
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Braz J Med Biol Res 31(11) 1998
F. Ribeiro-Dias et al.
activated macrophages kill YAC-1 cells re-mains unsettled.
Based on these observations, we asked whether mycoplasma-infected YAC-1 tumor cells could stimulate macrophages to release NO and if this molecule, in turn, will lyse the infected YAC-1 cells.
Six- to eight-week-old male BALB/c mice were used throughout the experiments. Mac-rophages were harvested by lavage of the peritoneal cavity with 5 ml of phosphate buffered saline (PBS) 5 days after intraperi-toneal injection of 1 ml of 3% thioglycollate medium (Gibco, Gaithersburg, MD). The cells were centrifuged and adjusted to 2 x 106 cells/ml in RPMI 1640 medium (Sigma
Chemical Co., St. Louis, MO) supplemented with 10 mM HEPES, 11 mM sodium bicar-bonate, 2 mM L-glutamine, 100 U/ml peni-cillin, 100 µg/ml streptomycin and 5% fetal bovine serum. The cell suspensions were plated (100 µl/well) onto 96-well flat-bot-tomed plates (Corning, New York, NY) and incubated for 2.5 h for adherence in a hu-midified atmosphere with 5% CO2 at 37oC.
The nonadherent cells were removed by washing the wells with PBS. The adherent cells were 80-90% macrophages as judged by neutral red uptake.
Mycoplasma-free YAC-1 tumor cells were cultured with supernatants from Myco-plasma arginini-infected cells (20% v/v) in an antibiotic-free medium. Infection was confirmed by culture of supernatants in SP4
medium (8). The infected cells were main-tained in complete medium. For cytotoxicity assays and induction of NO release, aliquots of tumor cell cultures were centrifuged, washed, resuspended in complete medium and adjusted to 5 x 105 cells/ml. Tumor cell
suspensions (used as target cells) were added to macrophage cultures (effector cells) at a 4:1 effector to target (E:T) ratio. Effector and target cells (at the same cell number/ well) were separately incubated in complete medium (100 µl/well) to establish control absorbance at 540 nm. The plates were incu-bated for 20 h in 5% CO2 in a humidified
atmosphere for cytotoxicity and NO release assays.
Macrophage anti-YAC-1 activity was evaluated by the MTT (3-(4,5-dimethylthi-azol-2yl)-2,5-diphenyltetrazolium bromide; Sigma) assay as described by Ferrari et al. (9). Briefly, 10 µl of MTT (5 mg/ml in PBS) was added to wells and the plates were incu-bated for 3 h. After this, 100 µl of 10% SDS in 0.01 N HCl was added in order to dissolve the formazan crystals generated by MTT reduction by living cells. The plates were incubated overnight and the absorbance was read at 540 nm in a microplate Labsystem Multiskan MS reader. Percent cytotoxicity was determined by the formula: % cytotox-icity = 1 - [(absorbance at 540 nm of effector + target cells) - (absorbance at 540 nm of effector cells)]/[absorbance at 540 nm of target cells] x 100.
NO release was determined by the stan-dard Griess reaction as follows: 50 µl of test solutions was added to 96-well flat-bottomed plates containing 50 µl/well of Griess re-agent (1% sulfanilamide/0.1% N-1-naphthyl-ethylene diamine dihydrochloride/2.5% H3PO4). After 10 min at room temperature
the absorbance of each well was measured in a Dynatech MR5000 microplate reader at 540 nm with a 620-nm reference filter and the nitrite concentration was determined from a sodium nitrite standard curve.
To prevent NO release we added 10 mM
µM Nitrite 123 123 123 123 % Cytotoxicity 80 70 60 50 40 30 20 10 0 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 * *
Noninfected YAC-1 cells Infected YAC-1 cells
Figure 1 - Mycoplasma arginini-infected YAC-1 tumor cells in-duce macrophage cytotoxic ac-tivity and nitric oxide production. Thioglycollate-elicited peritoneal macrophages (2 x 105) were co-cultured with YAC-1 tumor cells infected or not with
Myco-plasma arginini (5 x 104) for 20 h. Nitrite concentration was measured by the Griess reaction and macrophage cytotoxicity by the MTT assay. Results are re-ported as means ± SD for N = 4. *P<0.05 (Student t-test).
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L-NAME (Nω-L-arginine methyl ester; Sigma), an inhibitor of NO synthesis to the co-cultures. This concentration of L-NAME did not affect the viability of macrophages or tumor cells as evaluated by the MTT assay. Initially we compared NO production in co-cultures of thioglycollate-elicited macro-phages with YAC-1 cells infected or not with mycoplasma. As shown in Figure 1, macrophages co-cultured with mycoplasma-free YAC-1 cells produced very low levels of NO (4.7 ± 3.1 µM NO2-). Conversely,
high levels of NO (48.5 ± 13 µM of NO2-)
were detected in co-cultures with myco-plasma-infected YAC-1 cells. Figure 1 also shows that thioglycollate-elicited macrophag-es co-cultured with mycoplasma-free YAC-1 cells exhibited low cytotoxic activity (34.7 ± 8.6%). In contrast, macrophage cytotoxic activity increased significantly when tested against Mycoplasma arginini-infected YAC-1 cells (6YAC-1.4 ± 9.YAC-1%, P<0.05). Next, we asked whether inhibition of NO production by L-NAME would affect macrophage cyto-toxicity. As shown in Figure 2, addition of 10 mM L-NAME to macrophage co-cultures with mycoplasma-infected YAC-1 cells al-most completely inhibited NO release when compared with untreated co-cultures (51.3 ± 3 vs 3 ± 4 µM NO2
-). Most importantly, inhibition of NO production was paralleled by a significant decrease in macrophage cy-totoxicity (64.3 ± 4 vs 37.4 ± 2%).
Our results suggest that YAC-1 cells in-fected with Mycoplasma arginini stimulate macrophage NO production. In turn, NO exerts a cytotoxic activity against these tu-mor cells. However, it appears that YAC-1 cell killing does not rely entirely on NO release. This is documented in experiments where NO production was totally inhibited but significant cytotoxicity was still present (50%). Thus, it remains to be determined
whether macrophages are able to kill YAC-1 cells by an NO-independent mechanism or contaminating NK cells are operating in this situation. Mycoplasma-induced macrophage NO production has been previously docu-mented. For example, Mühlradt & Schade (10) found that murine resident peritoneal macrophages primed with interferon γ (IFNγ) released significant amounts of NO, when stimulated with a product extracted from Mycoplasma fermentans. Similarly, Yang et al. (11) showed that Mycoplasma arginini-infected L5178Y, a T-cell lymphoma, pro-duces a triggering factor for NO production by thioglycollate-elicited macrophages primed with IFNγ. Interestingly, in our ex-periments Mycoplasma arginini-infected tu-mor cells were able to induce NO production in thioglycollate-elicited macrophages in the absence of exogenous cytokines.
Since mycoplasmas are potentially asso-ciated with animal and human diseases in-cluding immunodeficiencies (12) it is pos-sible that macrophage activation induced by mycoplasma may contribute to clinical mani-festations of these disorders. Finally, myco-plasma frequently contaminates cell cultures (13), and thus may alter macrophage or other cell functions assayed in vitro.
µM Nitrite 12 12 12 12 % Cytotoxicity 80 70 60 50 40 30 20 10 0 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 Medium L-NAME * *
Figure 2 - Macrophage cytotoxic activity against Mycoplasma
ar-ginini-infected YAC-1 tumor
cells is partially mediated by ni-tric oxide. Thioglycollate-elicited macrophages (2 x 105) were co-cultured with Mycoplasma
argi-nini-infected YAC-1 tumor cells
(5 x 104) in the presence or ab-sence of 10 mM L-NAME for 20 h. Nitrite concentration was measured by the Griess reac-tion and macrophage cytotoxic-ity by the MTT assay. Results are reported as means ± SD for N = 4. *P<0.05 (Student t-test).
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